A reproducible method for biochemical, histological and functional assessment of the effects of ischaemia–reperfusion syndrome in the lower limbsIñig

[EN]Current methodology described to mimic lower limb ischaemia-reperfusion injury (LL-IRI) does not accurately define the procedures and pressures exerted to induce and maintain ischaemia. In this piece of work, we propose a well-defined and detailed rat model that simulates the conditions established in clinical practice guidelines for tourniquet application and allows us to test treatments that aim to prevent/reduce LL-IRI. Eighty-six male WAG/RijHsd rats were subjected to hind limb IRI (LL-IRI), using a mechanical system applying a 1 kg tension to induce and maintain ischemia for 2 or 3 h, and assessed the damage caused by reperfusion at biochemical and muscular levels at different time points. At the biochemical level, both 2 and 3 h of ischemia induced changes (except for electrolyte levels); 3 h of ischemia induced greater changes in specific markers of muscular damage: creatine kinase (CK) and lactate dehydrogenase (LDH). At the histopathological level, 3 h of ischemia and 24 h of reperfusion was associated with an increase in hind limb girth, cross-sectional area, and weight and presence of neutrophils, as well as histological damage in more than 60% of muscle fibres. Our model allows to reliably reproduce the damage associated with the use of a pneumatic tourniquet. CK and LDH, as well as measures of tissue damage, allow to define and characterize the response to LL-IRI-related damage. A period of 3 h of ischemia followed by 3 h of reperfusion caused only local damage but showed greater sensitivity to detect differences in future studies on prophylactic treatments against LL-IRI.No external funding was received. This study has been supported by internal funding from the Department of the Surgery, Radiology and Physical Medicine at the University of the Basque Country (UPV/EHU)

Curcumin Reduces Colorectal Cancer Cell Proliferation and Migration and Slows In Vivo Growth of Liver Metastases in Rats

Background: New therapeutic approaches are an essential need for patients suffering from colorectal cancer liver metastases. Curcumin, a well-known plant-derived polyphenol, has been shown to play a role in the modulation of multiple signaling pathways involved in the development and progression of certain cancer cells in vitro. This study aims to assess the anti-tumor effect of curcumin on CC531 colorectal cancer cells, both in vitro and in vivo. Methods: On CC531 cultures, the cell viability and cell migration capacity were analyzed (wound healing test) 24, 48, and 72 h after treatment with curcumin (15, 20, 25, or 30 µM). Additionally, in WAG/RijHsd tumor-bearing rats, the total and individual liver lobe tumor volume was quantified in untreated and curcumin-treated animals (200 mg/kg/day, oral). Furthermore, serum enzyme measurements (GOT, GPT, glucose, bilirubin, etc.) were carried out to assess the possible effects on the liver function. Results: In vitro studies showed curcumin’s greatest effects 48h after application, when all of the tested doses reduced cell proliferation by more than 30%. At 72 h, the highest doses of curcumin (25 and 30 µM) reduced cell viability to less than 50%. The wound healing test also showed that curcumin inhibits migration capacity. In vivo, curcumin slowed down the tumor volume of liver implants by 5.6-fold (7.98 ± 1.45 vs. 1.41 ± 1.33; p > 0.0001). Conclusions: Curcumin has shown an anti-tumor effect against liver implants from colorectal cancer, both in vitro and in vivo, in this experimental model

Partial hepatectomy enhances the growth of CC531 rat colorectal cancer cells both in vitro and in vivo

Partial hepatectomy (PHx) is the gold standard for the treatment of colorectal cancer liver metastases. However, after removing a substantial amount of hepatic tissue, growth factors are released to induce liver regeneration, which may promote the proliferation of liver micrometastases or circulating tumour cells still present in the patient. The aim of this study is to assess the effect of PHx on the growth of liver metastases induced by intrasplenic cell inoculation as well as on in vitro proliferation of the same cancer cell line. Liver tumours were induced in 18 WAG/RijHsd male rats, by seeding 250,000 syngeneic colorectal cancer cells (CC531) into the spleen. The left lateral lobe of the liver was mobilized and in half of the animals it was removed to achieve a 40% hepatectomy. Twenty-eight days after tumour induction, the animals were sacrificed and the liver was removed and sliced to assess the relative tumour surface area (RTSA%). CC531 cells were cultured in presence of foetal calf serum, non-hepatectomised (NRS) or hepatectomized rat serum (HRS), and their proliferation rate at 24, 48, and 72h was measured. RTSA% was significantly higher in animals which had undergone PHx than in the controls (non-hepatectomised) (46.98±8.76% vs. 18.73±5.65%; p<0.05). Analysing each lobe separately, this difference in favour of hepatectomized animals was relevant and statistically significant in the paramedian and caudate lobes. But in the right lobe the difference was scarce and not significant. In vitro, 2.5% HRS achieved stronger proliferative rates than the control cultures (10% FCS) or their equivalent of NRS. In this experimental model, a parallelism has been shown between the effect of PHx on the growth of colorectal cancer cells in the liver and the effect of the serum on those cells in vitro.This project has been partially funded by the Scientific Foundation of the Spanish Association Against Cancer (AECC) and the Basque Association for Surgical Research (AVEFIQ)

Antioxidant-Based Therapy Reduces Early-Stage Intestinal Ischemia-Reperfusion Injury in Rats

Intestinal ischemia-reperfusion injury (i-IRI) is a rare disorder with a high mortality rate, resulting from the loss of blood flow to an intestinal segment. Most of the damage is triggered by the restoration of flow and the arrival of cytokines and reactive oxygen species (ROS), among others. Inactivation of these molecules before tissue reperfusion could reduce intestinal damage. The aim of this work was to analyze the preventive effect of allopurinol and nitroindazole on intestinal mucosal damage after i-IRI. Wag/RijHsd rats were subjected to i-IRI by clamping the superior mesenteric artery (for 1 or 2 h) followed by a 30 min period of reperfusion. Histopathological intestinal damage (HID) was assessed by microscopic examination of histological sections obtained from injured intestine. HID was increased by almost 20% by doubling the ischemia time (from 1 to 2 h). Nitroindazole reduced HID in both the 1 and 2 h period of ischemia by approximately 30% and 60%, respectively (p < 0.001). Our preliminary results demonstrate that nitroindazole has a preventive/protective effect against tissue damage in the early stages of i-IRI. However, to better understand the molecular mechanisms underlying this phenomenon, further studies are needed.This research received funding from the University of The Basque Country UPV/EHU (grant reference GIU19/088)

Implementation of a Surgery Congress for Medical Students to Learn Transversal Competences. A Case of Student-Led Teaching Activity

Background: A dynamic training approach close to clinical work and research is highly requested by health sciences students. The aim of this paper is to present the organizational model of a student-promoted and student-managed surgical congress that encourages the acquisition of transversal competencies among the students in charge of the organization of the Congress. Methods: A two-day surgical congress for medical students organized by themselves was held. Each day comprised two separate sections corresponding to different surgical specialties; sections included three types of activities: conferences, round tables with guest professors, and practical workshops. Once the Congress had finished, an online survey was carried out to evaluate 10 items scored from 1 to 4. To assess the acquisition of transversal competences among the students organizing the congress, three evaluations were carried out by the professor involved in the organization of the congress. Results: The congress had great acceptance among the students, filling 150 available places with an attendance rate of 100%. The survey showed a high assessment of the subjects (3.48/4), conferences (3.48/4) and workshops (3.27/4). Evaluation of the round tables was significantly lower (2/4). A total of 99% considered the congress to have been useful in its formation process and 100% would recommend it. The grade of transversal competences among the students organizing the congress showed a significant increase between the first and the third evaluation, being between 1.24 and 7.25 times higher. Conclusions: the student-led student surgical congress is a well-evaluated activity for medical students, and promotes, among its organizers, the acquisition of transversal competences.The congress organization was partially supported by the Vice-rectorate of the Biscay Campus, the Student Council of the Faculty of Medicine and Nursing and the Student Council of the University of the Basque Country for their support

Conditioned Medium from H2O2-Preconditioned Human Adipose-Derived Stem Cells Ameliorates UVB-Induced Damage to Human Dermal Fibroblasts

Human skin exposure to ultraviolet B (UVB) radiation can result in acute photodamage through oxidative modifications of cellular components and biomolecules involved in the metabolism of dermal cells. Recently, the therapeutic potential of human adipose-derived stem cells (hASCs) has been investigated as a novel strategy for photoprotection due to their pro-angiogenic properties, protective activity against oxidative stress and paracrine effect on dermal cells. To enhance these therapeutic properties, hASCs can be preconditioned by exposing them to sublethal cellular stressors. In this study, we first analyzed response capacity against UVB-induced oxidative stress in H2O2-preconditioned hASCs (called HC016 cells); and second, we evaluated the photoprotective effect of HC016-conditioned medium (CM) in an in vitro UVB irradiation model in cultured human foreskin fibroblasts (hFFs). The results demonstrated that HC016 cells have a greater capacity to respond efficiently to UVB-induced oxidative stress, evidenced by higher Nrf2 antioxidant system activity and enhanced viability and migration capacity. Further, HC016-CM treatment increased viability, migratory capacity and collagen type I synthesis in hFFs exposed to UVB radiation, as well as reducing their cytotoxicity, apoptosis, senescence and IL-6 secretion. Collectively, these findings support the view that HC016 cells could protect against UVB-induced photodamage via paracrine mechanisms.This research was founded by the University of the Basque Country (UPV/EHU, grant reference numbers: GIU19/088 and PES 21/50)

Evolution of Culture on Patient Safety in the Clinical Setting of a Spanish Mutual Insurance Company: Observational Study between 2009 and 2017 Based on AHRQ Survey

Background: Patient safety (PS) is a key factor in reducing or even eradicating adverse incidents and events. Many health organizations promote strategies to improve PS, while also pointing out the importance of measuring it. For more than eight years, our institution has developed strategies focused on improving PS-culture among our personnel. The goal of this paper is to analyze the PS-culture between the years 2009 and 2017. Methods: A cross-sectional survey focused on PS, and developed by the American Agency for Healthcare Research and Quality (AHRQ), was conducted in 2009 and in 2017 among all healthcare workers at Mutualia, anonymously and voluntarily. Results: The overall response rate was similar in both 2009 and 2017 (37.2% and 38.5%, respectively). The average rating obtained showed a significant improvement over the period (7.7 vs. 8.1; p < 0.05). Itemizing by question, the main strengths were found in management support, organizational learning and continuous improvement, and, especially, in teamwork. Regarding weaknesses, the two lowest scores were those which refer to the balance between clinical safety and workload and the freedom to question the decisions made by superiors. Conclusions: The results obtained from the PS-surveys show that the overall PS-culture in our institution has increased, suggesting that the strategies focused on the improvement of PS-culture were well adopted among our personnel. The overall score places Mutualia at similar levels to those reached by the AHRQ and Spanish National Health System

Effects of Folinic Acid Administration on Lower Limb Ischemia/Reperfusion Injury in Rats

Surgery under ischemic conditions, lasting up to 3 h, is routinely performed in orthopedic surgery, causing undesirable injury due to ischemia-reperfusion syndrome, with short and medium-term functional repercussions. To date, there is no established prophylactic treatment. In this work we evaluated folinic acid (FA) in a rodent model of lower limb ischemia-reperfusion (IRI-LL). 36 male WAG rats underwent 3 h of lower limb ischemia. In the saline group, rats received intraperitoneal administration of saline (used as vehicle for treatment). In the experimental group, rats were pretreated with FA (2.5 mg/kg) before the end of ischemia. After ischemia, animals were sacrificed at 3 h, 24 h or 14 days (for biochemical determination (Na+, K+, Cl-, urea, creatinine, CK, LDH, ALP, ALT, and AST), pathological assessment, or functional study using the rotarod test; respectively). Another six animals were used to establish the reference values. The prophylactic administration of FA significantly reduced the elevation of biochemical markers, especially those that most directly indicate muscle damage (CK and LDH). In addition, it also improved direct tissue damage, both in terms of edema, weight, PMN infiltrate and percentage of damaged fibers. Finally, the administration of FA allowed the animals to equal baseline values in the rotarod test; what did not occur in the saline group, where pre-ischemia levels were not recovered. Following 3 h of lower limb ischemia, FA minimizes the increase of CK and LDH, as well as local edema and leukocyte infiltration, allowing a faster recovery of limb functionality. Therefore, it could be considered as a prophylactic treatment when tourniquet is used in clinics.This research received funding from the University of The Basque Country UPV/EHU (grant reference GIU19/088)

Folinic Acid Potentiates the Liver Regeneration Process after Selective Portal Vein Ligation in Rats

Liver resection remains the gold standard for hepatic metastases. The future liver remnant (FLR) and its functional status are two key points to consider before performing major liver resections, since patients with less than 25% FLR or a Child–Pugh B or C grade are not eligible for this procedure. Folinic acid (FA) is an essential agent in cell replication processes. Herein, we analyze the effect of FA as an enhancer of liver regeneration after selective portal vein ligation (PVL). Sixty-four male WAG/RijHsd rats were randomly distributed into eight groups: a control group and seven subjected to 50% PVL, by ligation of left portal branch. The treated animals received FA (2.5 m/kg), while the rest were given saline. After 36 h, 3 days or 7 days, liver tissue and blood samples were obtained. FA slightly but significantly increased FLR percentage (FLR%) on the 7th day (91.88 ± 0.61%) compared to control or saline-treated groups (86.72 ± 2.5 vs. 87 ± 3.33%; p < 0.01). The hepatocyte nuclear area was also increased both at 36 h and 7days with FA (61.55 ± 16.09 µm2, and 49.91 ± 15.38 µm2; p < 0.001). Finally, FA also improved liver function. In conclusion, FA has boosted liver regeneration assessed by FLR%, nuclear area size and restoration of liver function after PVL

The Prevention of Ischemia-Reperfusion Injury in Elderly Rats after Lower Limb Tourniquet Use

Background: Lower limb ischemia-reperfusion injury (IRI-LL) is a common major complication of orthopedic surgery, especially in elderly patients. It has previously been demonstrated that folinic acid (FA) reduced IRI-LL damage in 3–4-month-old rats. This current work analyses the effect of FA in the prevention of IRI-LL in elderly animals. Methods: Forty-two 18-month-old male WAG/RijHsd rats were subjected to 3 h of ischemia. Eighteen animals received FA (2.5 mg/kg, ip) 20 min before the end of the ischemia period, while the other half received the same volume of saline solution. The animals were sacrificed after 3 h, 24 h, and 14 days of reperfusion for biochemical (tissue damage markers and electrolytes), histopathological studies of the gastrocnemius muscle and the daily assessment of the limb function by the Rota Rod test, respectively. Results: The administration of FA prior to the end of the ischemia period reduced the increase in LDH and CK observed in non-treated animals by 30–40% (p < 0.0001). When the histological sections were analyzed, FA was found to have reduced the number of damaged muscle fibers per field by 20% (60 ± 17.1 vs. 80.7 ± 16.4, p < 0.0001). The functional test revealed that FA also led to an improvement in the muscle function, assessed by the length of time that the animals kept running on the rod, compared to untreated animals. Conclusions: The administration of FA, prior to the end of the ischemic period, decreases the damage induced by IRI-LL, also achieving a faster recovery of mobility.This research was funded by the University of the Basque Country UPV/EHU (grant reference GIU19/088)